Difference between revisions of "Fragment Purification"
From GcatWiki
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[http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel | [http://www.mn-net.com/tabid/1452/default.aspx Nucleospin Extract II]kit from Machery-Nagel | ||
| − | + | #With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube. | |
| − | #With a plastic ruler | + | #Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears. |
| − | # | + | #Incubate a dH20 in 55 C water bath for at later use. |
| − | + | #Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed | |
| − | + | #Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds. | |
| − | #Transfer | + | #Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column, then spin 30 seconds. |
| − | #Dump the collection tube, transfer the remaining volume to the column, and spin | + | #Repeat step 6. |
| − | #Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column | + | #Dump collection tube and add 250 ul wash buffer NT3 to column, then spin 30 seconds. |
| − | + | #Repeat step 8. | |
| − | #Repeat | + | #Dump the collection tube and spin the column at full speed for 1 minute to dry colum. |
| − | #Dump collection tube and add 250 ul wash buffer NT3 to column | + | #Transfer to a NEW 1.5 ml tube. |
| − | + | #Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds. | |
| − | #Repeat | + | #Reapply the eluate onto the column, let stand 1 minute, spin 1 minute. |
| − | + | #Measure concentration on Nanodrop | |
| − | #Dump the collection tube and spin the column at full speed for 1 minute | ||
| − | #Transfer | ||
| − | #Add | ||
| − | |||
| − | #Reapply the eluate onto the column | ||
| − | |||
| − | #Measure concentration on Nanodrop | ||
Latest revision as of 14:13, 19 May 2015
Fragment Purification from an Agarose Gel
Nucleospin Extract IIkit from Machery-Nagel
- With a plastic ruler cut band out of agarose gel and place in 1.5 microcentrifuge tube.
- Add 700 ul of buffer NTI and incubate at least 5 minutes in 55 C water bath, vortexing every minute until gel slice disappears.
- Incubate a dH20 in 55 C water bath for at later use.
- Transfer 700 ul to a Nucleospin Extract II column (NOT a miniprep column) on a collection tube and spin 30 seconds at full speed
- Dump the collection tube, transfer the remaining volume to the column, and spin 30 seconds.
- Dump collection tube and add 700 ul wash buffer NT3 (make sure ethanol has been added to the NT3 stock) to column, then spin 30 seconds.
- Repeat step 6.
- Dump collection tube and add 250 ul wash buffer NT3 to column, then spin 30 seconds.
- Repeat step 8.
- Dump the collection tube and spin the column at full speed for 1 minute to dry colum.
- Transfer to a NEW 1.5 ml tube.
- Add 12 ul heated dH2O directly to filter, let stand 30 seconds, and spin 30 seconds.
- Reapply the eluate onto the column, let stand 1 minute, spin 1 minute.
- Measure concentration on Nanodrop
